Revista Romn de Medicin de Laborator Vol. 18, Nr.

2/4, Iunie 2010

77

COURSE NOTES
Laboratory diagnosis of acute leukemia
Diagnosticul de laborator al leucemiilor acute
Selicean I. Elena-Cristina*, Paiu Mariana
Institute of Oncology Prof. dr. I. Chiricu Cluj-Napoca,
Medical Laboratory Hematology

Abstract
Acute leukemias are a heterogeneous group of hematopoietic malignancies, characterized by uncontrolled growth of a leukemic clone, which is blocked in the maturation process. First classifications of acute leukemias were based solely on morphologic criteria, but the actual trend is to take into account biological and
pathogenetic aspects. This review's purpose is to cover this complex subject, according to the latest WHO classification, in respect with examination requirements and from the perspective of laboratory physicians.
Keywords: acute leukemia, morphology, immunophenotyping, cytogenetics, molecular biology

Rezumat
Leucemiile acute reprezint un grup heterogen de mbolnviri acute ale sistemului hematopoietic, caracterizate prin proliferarea necontrolatra a unei clone de celule leucemice blocat in maturaie. Dac primele
clasificri ale LA se bazau exclusiv pe criterii morfologice, tendina actual de abordare vizeaz aspecte biologice si patogenetice. Lucrarea i propune abordarea acestui subiect complex, respectnd cerinele tematicilor de
examen pentru medici rezideni si specialiti, innd cont de clasificarea actual a OMS i abordnd diagnosticul
LA din perspectiva medicului de laborator.
Cuvinte cheie: leucemie acut, morfologie, imunofenotipare, citogenetic, biologie molecular

Corresponding author: Selicean Elena-Cristina,

Tel. 0723 769479, E-mail: cristinaselicean@hotmail.com

78

Revista Romn de Medicin de Laborator Vol. 18, Nr. 2/4, Iunie 2010

Definition, mechanisms, terminology for AL

Definiie, mecanisme i terminologie n LA

Acute leukemias are a heterogeneous

group of hematopoietic system malignancies,
characterized by uncontrolled growth of a cell
clone which does not mature normally replaces
normal hematopoietic tissue and can invade extramedular sites.
Proliferation may occur from a myeloid
or lymphoid cell. ALs are classified into two
broad categories: myeloid and lymphoblastic.
The term of myeloid leukemia includes
proliferation of granulocytic, monocytic,
erythroid, megakaryocytic lineage, referred as
non-lymphoid leukemias.
Patients with AL present anemia, thrombocytopenia, usually neutropenia and blasts in
peripheral blood.

Leucemiile acute sunt un grup heterogen

de boli maligne ale sistemului hematopoietic,
caracterizate prin proliferarea necontrolat a
unei clone de celule imature, blocate n maturaie, care nlocuiete esutul hematopoietic normal i poate invada teritorii extramedulare.
Proliferarea se poate produce pornind de
la o celul mieloid sau limfoid, LA fiind clasificate n dou mari categorii: mieloblastice i
limfoblastice.
Termenul de LA mieloblastic include proliferri de linie mieloid granulocitar, monocitar,
eritrocitar, megakariocitar, termenul alternativ pentru denumirea lor comun fiind LA non-limfoide.
Pacienii cu LA se prezinta cu anemie ,
trombocitopenie, de obicei neutropenie i blati
n sngele periferic.

FAB classification of AL

Clasificarea FAB a LA

The first classification of AL based

solely on morphological criteria was made in
1976 by a group of experts of the French-American-British group, and was later revised.
The latest FAB classification, released in
1985, includes 8 types of AML and 3 types of ALL.
The main diagnostic criterion is quantitative: over 30% blasts of the total nucleated
non-erythroid cells in bone marrow smear examined under a microscope in Romanovski
(May-Grnwald Giemsa) stain. The next step in
the FAB classification is to establish lineage assignment of the blasts by using morphological
criteria and cytochemical staining.
The FAB classification of AML is based
on the type of myeloid series that proliferates and
the degree of maturation of the leukemic clone.
Thus, acute myeloid leukemia without
maturation (M1), acute myeloid leukemia with
maturation (M2) and acute promyelocytic leukemia (M3) are proliferations of the granulocytic
series, with different degrees of maturation.
For all other FAB types, their name in-

Prima clasificare a LA, bazat exclusiv

pe criterii morfologice, a fost fcut de un grup
de experi Franco-Americano-Britanic n 1976,
ulterior revizuit.
Ultima versiune a clasificrii FAB (1985)
include 8 tipuri de LAM i 3 tipuri de LAL.
Principalul criteriu de diagnostic este
cantitativ: peste 30% blati din totalul de elemente nucleate non-eritroide n aspiratul medular examinat la microscop, n coloraie tip Romanovski (May-Grnwald Giemsa). Urmtoarea
etapa n vederea clasificrii pe criterii FAB este
stabilirea apartenenei de linie a blatilor, tot pe
criterii morfologice, eventual folosind coloraii
citochimice ajuttoare.
Clasificarea FAB a LAM se face n
funcie de seria/seriile mieloide care prolifereaz
i gradul de maturaie a clonei leucemice.
Astfel, leucemia acuta mieloid fr maturaie (M1), leucemia acuta mieloid cu maturaie (M2) i leucemia acuta promielocitar
(M3) sunt proliferri de serie granulocitar, cu
grade de maturaie diferite.
i pentru celelalte tipuri FAB denumirea

Revista Romn de Medicin de Laborator Vol. 18, Nr. 2/4, Iunie 2010

dicates lineage assignment: M4- myelomonocytic, M5-monocytic (M5a-monoblastic without

maturation and M5b monoblastic with maturation), M6-erythroleukemia, M7-megakaryoblastic leukemia, M0 - acute myeloid leukemia
without differentiation (Table 1).
FAB group classifies LAL in three categories, based upon the morphological appearance of blasts (Table 2).
L1 and L2 are similar in terms of morphology and it has been proved that they do not
represent clinically and biologically distinct entities. L3 has a morphological appearance highly
suggestive of lymphoblastic leukemia- Burkitts
lymphoma, which requires confirmation by
demonstrating a mature B phenotype.

79

indic apartenena de serie a clonei leucemice:

M4 - este leucemia acuta mielomonocitar, M5 leucemia acuta monocitar (M5a monoblastic
fr maturaie i M5b - monoblastic cu maturaie), M6 - eritroleucemie, M7 - leucemie acut
megakarioblastic, M0 - leucemie acut mieloid minim difereniat (Tabelul 1).
Grupul FAB clasific LAL, pe baza aspectului morfologic al blatilor, n trei categorii
(Tabelul 2).
Categoriile L1 i L2 sunt asemntoare
din punct de vedere morfologic. S-a dovedit c nu
reprezint nici din punct de vedere clinic i biologic entitti distincte. L3 reprezint aspectul morfologic nalt sugestiv pentru leucemia limfoblastic limfomul limfoblastic Burkitt, care necesit confirmare prin imunofenotipare (fenotip B-matur).

Table 1. FAB classification of AML

FAB

Blasts in BM

Granulocytic
component

Monocytic
component

Other

M1

90% from NEC,

3% MPO+

10% from NEC

M2

30-89% from NEC

>10% from NEC

<20% from NEC

M3
20% from NEC
(including
myeloblasts)
M4

Typical morphology

30% from NEC

(myeloblasts,
monoblasts and
promonocytes)

20% fron NEC

Similar aspect with M2

Additional diagnostic criteria :

-monocytes in PB>=5x 10 9/l
-seric lysozyme raised
-cytochemistry- specific aspect
Additional criteria :
-monocytes in PB >=5x 10 9/l and
seric/ urinary lysozyme three times
normal values
-monocytes in PB>=5x 10 9/l and
cytochemistry specific for monocytic
series

M5a

Monoblasts 80% from

the monocytic series

80% from NEC

M5b

Monoblasts<=80% from
the monocytic series

80% from NEC

30% from NEC

Erythroblasts >= 50%

lineage assignment to megakaryocytes

documented by ultrastructural
cytochemistry (platelet peroxydase) or
by immunophenotyping
(megakaryocytic markers)

M5

M6

M7

M0

<3% MPO+

Myeloid immunophenotype

80

Revista Romn de Medicin de Laborator Vol. 18, Nr. 2/4, Iunie 2010

Table 2. FAB classification of ALL

Blasts

Cytoplasm

L1

small

scant

L2

medium

L3

large

moderate,
week basophilic
Abundant,
intense basophilic,
sometimes vacuolated

Nucleus
Rather coarse chromatin,
Nucleoli - indistinct or absent
Finely dispersed, evident nucleoli
condensed
variable visible nucleoli

Identification of blasts - morphological

criteria

Identificarea blatilor - criterii

morfologice

The percentage of blasts in bone marrow is determined by counting 500 nucleated

elements.
FAB Group recognizes 3 types of blasts
with distinct morphological characters: lymphoblast, type I agranular myeloblasts and type II
myeloblasts with azurophilic granules. National
Cancer Institute Workshop describes an additional type III myeloblast with more than 20
azurophilic granules in the cytoplasm.
From a practical perspective and as recommended by the International Working Group
on Morphology, it is important to describe the
blasts as agranular and granular.
Agranular blasts are seen in MPO-negative acute leukemias: ALL, AML-M0, M6, M7.
Today it is considered that classical morphology
yields only indicative criteria for identifying
these types of blasts and ultrastructural studies
and immunophenotyping are needed for their
correct classification.
Regarding granular blasts, it is important to differentiate them from normal and dysplastic promyelocytes. The main difference
between a granular blast and a normally appearing
promyelocyte is the clear perinuclear hof (Golgi
area), a promyelocytic feature. An exception to
this rule are the granular blasts of AML with
t(8; 21), which may have a separate small Golgi
area, but no other characters of a promyelocyte.

Determinarea procentului de blati n

mduva osoas se face prin numrarea a 500
elemente nucleate.
Grupul FAB recunoate 3 tipuri de blati cu
caractere morfologice distincte: limfoblast, mieloblast de tip I - agranular i mieloblast de tip II - cu
granulaii azurofile. National Cancer Institute Workshop descrie un tip adiional, mieloblastul de tip III
cu peste 20 granulaii azurofile n citoplasm.
Din punct de vedere practic i conform
recomandrilor International Working Group on
Morphology este important descrierea blatilor
ca agranulari i granulari.
Blatii agranulari se ntlnesc n leucemiile acute MPO-negative: LAL, LAM - M0,
M6 i M7. n prezent se consider c morfologia
clasic ofer doar criterii orientative de identificare a acestor tipuri de blati i c sunt necesare
studii de ultrastructur i imunofenotip pentru
corecta lor ncadrare.
n ceea ce privete mieloblatii granulari,
este important diferenierea lor de promielocitul
normal i displazic. Principala deosebire a blatilor granulari de promielocitul normal este considerat zona clar perinuclear (zona Golgi) caracteristic promielocitului. Excepia de la aceast
regul o reprezint blatii granulari din LAM cu
t(8;21), care pot avea o mic zon Golgi distinct,
dar fr alte caractere de promielocit.
Promielocitul displazic ntrunete, pe

Revista Romn de Medicin de Laborator Vol. 18, Nr. 2/4, Iunie 2010

81

Table 3. Morphological aspects of monocytic lineage

Nucleus

Chromatin

Cytoplasm

Other

Monoblast

Round/ oval

delicate/ lacy
evident nucleolus

Basophilic, rare azurophilic granules

large, 20-30m

Promonocyte

Convoluted/
indented

delicate/ lacy
evident nucleolus

Variable basophilia,variable azurophilic Monoblast- like, but

granules
different nucleus shape

Immature
monocyt e

Convoluted/
indented

condensed,
sometimes
nucleolated

Intermediate basophilia between

promonocytes or blasts and mature
monocytes

Monocyte

Lobulated/
indented

Condensed, without Grey, occasionally azurophilic granules,

large, 20-25m
nucleoli
vacuoles

Dysplastic promyelocytes display features belonging to the maturation stage, in addition to abnormal patterns: uneven cytoplasmic
basophilia, poorly represented Golgi zone,
hipergranularity or hipogranularity, irregular distribution of granules.
The morphological identification of
these forms is important for:
MDS-differential diagnosis - where the percentage of blasts is crucial
differential diagnosis of AML - agranulocytosismorphological aspects can be critical in certain
stages
further laboratory investigation for a possible
t(8,21).
In AML-M3 malignant promyelocytes
are counted as blasts. In the classical form, they
are hypergranular and may have multiple Auer
rods. The morphologic microgranular/ hypogranular variant requires cytogenetic confirmation (the presence of t(15, 17)). Prompt diagnosis of APL is followed by establishment of specific therapy and DIC prevention.
Other situations in which morphological
assessment of blasts is crucial for diagnosis are
proliferations of the monocytic compartment. In
these cases the percentage of blasts results by
adding the following: myeloid blasts, monoblasts and promonocytes. Differential diagnosis
of chronic versus acute myelomonocytic leukemia depends on the morphological differentiation of monoblasts and promonocytes from
more mature monocytes (Table 3).

Resembles monocytes, but

more immature and smaller

lng caracterele aparinnd stadiului de maturaie, aspecte anormale: bazofilie citoplasmatic

neuniform, zona Golgi slab reprezentat, hipergranularitate sau hipogranularitate, distribuie
neregulat (n grmezi) a granulaiilor.
Identificarea acestor forme morfologice
este important pentru:
diagnosticul diferenial LAM - SMD - unde
procentul de blati este hotrtor
diagnosticul diferential LAM - agranulocitoz,
unde aspectele morfologice pot fi decisive n
anumite stadii
orientarea investigaiilor n vederea eventualei
identificri a t(8;21).
n LAM-M3, promielocitele maligne sunt
numrate ca i blati. n forma clasic acestea sunt
hipergranulare i pot prezenta corpi Auer multipli
dispui n grmad de surcele. Varianta morfologic microgranular/hipogranular necesit confirmare citogenetic (prezena t(15;17)). Diagnosticul
pozitiv de LA promielocitar este decisiv pentru instituirea terapiei specifice i poate reprezenta o urgena din cauza riscului crescut de CID.
O alt situaie n care evaluarea morfologic a blatilor este crucial pentru diagnostic sunt
proliferrile de linie sau cu component monocitar. n aceste cazuri procentul de blati este reprezentat, n funcie de situaie, de nsumarea procentelor urmtoarelor elemente: mieloblati, monoblati i promonocite. Diagnosticul diferenial al
LA de leucemia mielomonocitar cronic depinde
de deosebirea morfologic a monoblatilor i promonocitelor de monocitele mai mature (Tabelul 3).

82

Revista Romn de Medicin de Laborator Vol. 18, Nr. 2/4, Iunie 2010

Other important morphological characters for the diagnosis and classification of AML
are dysplastic changes of hematopoiesis. Thus,
the identification of more than 50% dysplastic
elements in at least two myeloid lineages allows
categorization, according to recent WHO criteria, in the category of dysplasia related AML.
Observation of abnormal eosinophils with large,
basophilic granules guides investigations towards identification of inv(16) or t(16,16).

Alte caractere morfologice importante pentru diagnosticul i clasificarea LA sunt reprezentate

de modificrile displazice ale seriilor mieloide. Astfel, identificarea a peste 50% elemente displazice n
cel puin dou linii mieloide permite ncadrarea,
conform criteriilor recente ale OMS, n categoria de
LAM legat de displazie, iar evidenierea pe frotiu a
unor eozinofile anormale, cu granulaii mari, bazofile, orienteaz investigaiile n vederea identificrii
inv(16) sau t(16;16).

Cytochemical stains improve accuracy

and reproducibility of lineage assignment in
AL. The most useful cytochemical stains are
peroxidase and non-specific esterase.
Myeloperoxidase is present in granulocytic and monocytic cells, absent in lymphocytic
cells. Therefore MPO-stained myeloid cells
(from granular blasts, promyelocytes to segmented granulocytes) have a specific yellowish-brown color, proportional to the content of
MPO; monocytes are weakly positive; lymphocytic cells are always negative. Interpretation requires experience in order to distinguish false
negative reactions (for technical reasons) from
situations with peroxidase deficiency (dysplastic
feature sometimes encountered in AML).
As per the FAB classification, peroxidase
reaction allows differentiation in POX- positive
and POX-negative AL and differentiation of AMLM0 and AML-M1 (the percentage of POX-positive blasts), aids the identification of the monocytic
component by specific reaction appearance (thin
veil) and detects the presence of Auer rods more
frequently than the MGG stain.
In most laboratories where immunophenotyping is available, cytochemical stains
were abandoned.
The Perls reaction can be useful in AL
for identification of ringed syderoblasts as a dysplastic feature.
The FAB classification represents the first
attempt to divide the heterogeneous group of acute
leukemias, where each of the subtypes does not represent a distinct clinical and biological entity.

Coloraiile citochimice mbuntesc

acurateea i reproductibilitatea identificrii de
linie n LA. Cele mai utile coloraii citochimice
sunt mieloperoxidaza i esteraza nespecific.
Mieloperoxidaza este o enzim din granulaiile celulelor de linie granulocitar i monocitar, absent n celulele de linie limfocitar. n
consecint celulele de linie granulocitar prezint n coloraia specific, o reacie glbui-maronie
proporional cu coninutul de MPO; monocitele
sunt slab pozitive; celulele de linie limfocitar
sunt ntotdeauna negative. Interpretarea reactiei
mieloperoxidazei necesit experien pentru a
putea distinge reaciile fals negative (din motive
tehnice) de situaiile cu deficit de peroxidaz
(caracter displazic ntlnit uneori n LAM).
Reacia mieloperoxidazei permite diferenierea LA n MPO - pozitive i MPO - negative i
diferenierea LAM - M0 de LAM - M1 (prin procentul de blati MPO - pozitivi), ajut la identificarea componentei monocitare prin aspectul specific
al coloraiei (vl fin) i detecteaz mai frecvent dect coloraia MGG prezena corpilor Auer.
n majoritatea laboratoarelor n care este
disponibil imunofenotiparea, reaciile citochimice au fost abandonate.
In LA mai poate fi util coloratia cu albastru de Prusia - reacia Perls pentru identificarea sideroblatilor inelari ca semn de displazie.
Clasificarea FAB a LA reprezint prima
ncercare de mprire a grupului heterogen al
leucemiilor acute, fr ca fiecare din subtipuri s
reprezinte o entitate distinct din punct de vedere clinic i biologic.

Revista Romn de Medicin de Laborator Vol. 18, Nr. 2/4, Iunie 2010

Subsequently, the classification proposed by MIC Cooperative Study Group (morphology, immunology, cytogenetics) paved the
way for the recognition of the significance of
cytogenetics and immunophenotype in AL.
The next classification proposed and
widely accepted was the WHO classification of
2001, revised in 2008. The principles underlying
this classification are:
the percentage of blasts required for diagnosis
according to WHO criteria is 20%
the percentage of blasts is not a diagnostic criterion
for AL, when there is evidence of recurrent cytogenetic mutations in a significant hematologic context
includes two provisional entities characterized
by mutations in NPM1 and CEBPA
introduces MDS- related AML, diagnosis based
on MDS history, cytogenetic abnormalities associated with MDS, multilineage dysplasia
introduces the post-chemotherapy AML class
maintains FAB terminology for morphological
classification of AML in the category "not otherwise specified"

83

Ulterior, clasificarea propus de Grupul

de Studiu Cooperativ MIC (morfologie, imunologie i citogenetic) a deschis calea pentru recunoaterea semnificaiei modificrilor citogenetice i respectiv a imunofenotipului n LA.
Urmtoarea clasificare propus i acceptat pe scar larg a fost clasificarea OMS din
2001, revizuit n 2008. Principiile pe care se
bazeaz aceast clasificare sunt urmtoarele:
procentul de blati necesar pentru diagnosticul
pozitiv de LA este de 20%;
procentul de blati nu mai reprezint criteriu
de diagnostic pentru LA atunci cnd ntr-un
context hematologic semnificativ se demonstreaz prezena unei mutaii citogenetice recurente;
include dou entiti provizorii caracterizate
prin mutaii NPM1 i CEBPA;
introduce clasa LAM legat de SMD, avnd
ca i criterii de diagnostic antecedentele de
SMD, anomaliile citogenetice asociate SMD,
displazia multilinial;
introduce clasa LAM post- chimioterapie;

WHO 2008 classification of AL

I.
I.1

I.2
I.3
I.4
I.5
.

Acute myeloid leukemia

AML with recurrent cytogenetic abnormalities
I.1.1
AML with t(8:21)(q22;q22)
I.1.2
AML with inv(16)(p13.1q22) or t(16;16)
(p13.1;q22)
I.1.3
AML with t(15;17)(q22;q12)
I.1.4
AML with t(9;11)(p22;q23)
I.1.5
AML with t(6;9)(p23;q34);
I.1.6
AML with inv(3)(q21;q26.2) or t(3;3)
(q21;q26);
I.1.7
AML with t(1;22)(p13;q13)
AML with mutated NPM1
Provisional entities :
AML with mutated CEBPA
AML dysplasia related
Myeloid neoplasia therapy related
Myeloid sarcoma
Myeloid proliferations Down syndrom related
I.5.1 Transitory abnormal myelopoiesis
I.5.2 AML Down syndrome associated

(RUNX1;RUNX1T1)
(FBMYH11)
(PML-RARA)
(MLLT3-MLL)
(DEK-NUP214)
(RNP1-EVI1)

(promyelocytic)

(RMB15-MKL1)

(megakaryoblastic)

84

I.6

II.

III.

Revista Romn de Medicin de Laborator Vol. 18, Nr. 2/4, Iunie 2010

AML not otherwise specified

I.6.1 AML with minimal differentiation
I.6.2 AML without maturation
I.6.3 AML with maturation
I.6.4 Myelomnocytic AL
I.6.5 Myeloblastic/ monocytic AL
I.6.6 Acute erythroid leukemia
Pure erythroid leukemia
Erythroleukemia (erythroid/ myeloid)
I.6.7 Acute megakaryoblastic leukemia
I.6.8 Acute basophilic leukemia
I.6.9 Acute panmyelosis with myelofibrosis
Acute leukemia of ambiguous lineage
II.1
AL undifferentiated
II.2
AL mixed phenotype
with t (9;22)(q34;q11.2)
BCR-ABL1
II.3
AL mixed phenotype
with t(v;11q23);
MLL rearrangements
II.4
AL mixed phenotype
B/myeloid, NOS
II.5
AL mixed phenotype
T/myeloid NOS
II.6
AL mixed phenotype
NOS-rare types
II.7
Acute lymphoblastic leukemia/ lymphoma NK cells
Acute lymphoblastic leukemia/ lymphoma
III.1
ALL/ LL B NOS
III.2
ALL/LL B with recurrent cytogenetic abnormalities
III.2.1 t (9;22)(q34;q11.2)
BCR-ABL1
III.2.2 t(v;11q23)
MLL rearrangements
III.2.3 (12;21)(p13;q22)
TEL-AML1 (ETV6-RUNX1)
III.2.4 hyperdiployd
III.2.5 hypodiployd t(5;14)(q31;q32);
IL3-IGH
III.2.6 t(1;19)(q23;p13.3)
E2A-PBX1 (TCF3-PBX1)
III.3
ALL/LL T

classifies

ALL and ambiguous line leukemias

on immunophenotypic and genetic criteria
correlates ALL with lymphoblastic lymphoma.

Imunophenotyping - role in diagnosis of AL

Technical Considerations
Determination of cell surface antigen
expression may be done on cytological and histological preparations (imunocytochemistry,
imunohistochemistry), but imunophenotyping
by flow-cytometry became the most widely used
because it has many advantages.

menine

terminologia FAB pentru clasificarea

morfologic a LAM din categoria not otherwise specified ;
clasific LA limfoblastice i de linie ambigu
pe criterii de imunofenotipare i genetic;
coreleaz LAL cu echivalentul tisular de limfom limfoblastic.

Imunofenotiparea rolul n diagnosticul LA

Consideraii tehnice
Determinarea expresiei antigenelor de
suprafa celular se poate face pe preparate cito-

Revista Romn de Medicin de Laborator Vol. 18, Nr. 2/4, Iunie 2010

Currently available protocols for in vitro

diagnostic allow concurrent reading at up to 3-4
wavelengths and perform a multiparametric, objective, sensitive and rapid analysis of a large
number of cells, with the ability to delineate and
characterize distinct populations of cells in the
analyzed sample in terms of expression of surface and intracellular antigens.
This technique may be used in samples
of peripheral blood, bone marrow, and when
warranted, cerebrospinal fluid, other fluids
(pleural, pericardic, peritoneal), tumor masses.
Before being analyzed, the primary
sample is subject to a preliminary process for
separating mononucleate cells in a concentration
gradient or for red cell lysis. Many laboratories
prefer lysing protocols, because separation
methods are more laborious and cells could get
lost during processing. For analysis of intracellular antigens the method involves a step consisting in permeabilisation of cell membranes.
The current strategy for demarcating cell
populations from the sample ("gating) is plotting
CD45 versus SSC (side scatter - side scattering of
a laser beam). Blasts have a low side scatter (because they are weak granular) and express low
CD45 +, compared to other nucleated cells in the
sample: lymphocytes intensely CD45 +, erythroblasts CD45 negative, precursors of granulocytic
series high side scatter.
Antibody panel selection
The use of minimal antibody panels for
diagnostic purposes is recommended, since they
offer great diagnostic information and are reproducible in laboratories with different equipment.
The panel should help distinguishing
between AML and ALL, establishing lineage assignment (myeloid, monocytic, megakaryocytic,
erythroid, namely B or T), highlighting possible
aberrant expression of antibodies and outlining a
characteristic immunophenotype (leukemia-associated immunophenotype "LAIP")
The choice of fluorochrome depends on the
type of device; most devices can use fluorescein isothiocyanate, phycoerythrin, phycoerythrin-cyanin 5.

85

logice (imunocitochimie) sau histologice (imunohistochimie), dar n practic s-a impus imunofenotiparea prin citometrie n flux continuu, datorit multiplelor avantaje pe care le prezint.
Metodele aplicate n prezent n scop
diagnostic permit citirea concomitent la 3-4
lungimi de und i efectueaz o analiz multiparametric, sensibil i rapid a unui numr mare
de celule, oferind posibilitatea de a delimita i
caracteriza din punct de vedere al expresiei antigenelor de suprafa i intracelulare, populaiile
celulare din eantionul analizat.
Tehnica se poate aplica pe probe de snge
periferic, mduv osoas, iar atunci cnd este justificat, lichid cefalorahidian, alte lichide (pleural,
pericardic sau peritoneal) sau mase tumorale.
nainte de a fi analizat, proba primar se
supune unui procedeu de separare a mononucleatelor n gradient de concentraie sau unui procedeu de
liz a hematiilor. Multe laboratoare prefer metoda
cu liz, deoarece metodele de separare sunt mai laborioase i exist riscul de a pierde celule pe parcursul procesului de prelucrare. Pentru analiza antigenelor intracelulare, metoda presupune i o etap de
permeabilizare a membranei celulare.
Strategia actual pentru delimitarea populaiilor de celule din proba de analizat (strategia de
gating), pentru leucemiile acute, este CD45 versus
SSC (side scatter- dispersia laterala sub unghiuri mari
90o a fasciculului laser). Blatii au un grad redus de
dispersie laterala a fascicului (deoarece sunt slab granulari) i exprim CD45 dim+. Aceasta permite diferenierea lor de celelalte celule nucleate din proba
analizat: limfocite CD45 +++, eritroblati CD45-,
precursori neutrofilici i eozinofilici intensitate
mare a fasciculului laser dispersat lateral.
Alegerea panelului de anticorpi
n practic se urmrete utilizarea unor
paneluri minimale de anticorpi, care s permit
obinerea unor date cu valoare diagnostic mare
i reproductibile n laboratoare cu dotri diferite.
Panelul trebuie s permit deosebirea LAM
de LAL, stabilirea apartenenei la o linie (mieloid,
monocitar, megakariocitar, eritrocitar, respectiv B
sau T), evidenierea expresiei aberante de anticorpi,

86

Revista Romn de Medicin de Laborator Vol. 18, Nr. 2/4, Iunie 2010

Interpretation
For practical reasons, it was established
that the positivity limit is 20% for surface antigens, and 10% for cytoplasmic markers.
Differentiation of ALL AML
AML blasts express immature neutrophilic and monocytic antigens: CD13, CD15,
CD33, CD64, CD117, MPO.
The most sensitive and specific marker
for B lymphoblasts is CD19, followed by
CD22c. In T lineage lymphoblasts the highest
sensitivity is provided by CD3c.
Immunological characterization of
POX-negative AML
Immunophenotyping is essential for differential diagnosis of POX- negative ALs.
Expression of CD13, CD33 and MPO,
together with the lack of expression of CD19,
CD79a, TdT and CD3c demonstrates an AML
phenotype.
AML-M0 is defined as having less than
3% MPO-positive blasts in classic cytochemistry. The presence of MPO in turn can be better
demonstrated by immunophenotyping and electron microscopy. In these conditions, at least one
positive myeloid marker (CD113, CD33, or
MPO), in the absence of lymphoid markers
(CD3, CD22a, Cd79a) and megakaryocytic
markers (CD41 and CD61), allows the diagnosis
of AML-M0. HLA-DR, CD34, CD117, CD65,
CD14 and CD15 may be positive and sometimes
lymphoid markers (CD2, CD4, CD7, CD19) are
aberrantly expressed by myeloid cells.
Blasts of AML-M7 express megakaryocytic markers: CD41, CD42, CD61.
In erythroleukemia blasts express
CD235 or the characteristic phenotype CD36 + /
CD64-/MPO-.
Immunological characterization of AML morphological classes
AML-M1 and AML-M2 are characterized by the expression of CD13, CD33, MPO,
CD34, and CD117, without expression of
CD79a, CD14, CD64.

care s contureze un imunofenotip asociat leucemiei.

Alegerea fluorocromilor depinde de tipul aparatului,
majoritatea aparatelor pot utiliza izotiocianat de fluorescein, ficoeritrin, ficoeritrin - cyanin 5.
Interpretare
Din raiuni practice s-a stabilit ca limit
de pozitivitate expresia la peste 20% din celulele
populaiei examinate a antigenelor de suprafa,
respectiv 10% pentru markeri citoplasmatici.
Diferenierea LAM de LAL
n principiu, blatii din LAM exprim antigene imature de linie neutrofilica i /sau monocitar: CD13, CD15, CD33, CD64, CD117 i MPO.
Markerul cel mai sensibil i specific
pentru limfoblatii de linie B este CD19, urmat
de CD22c. Pentru limfoblatii de linie T, cea mai
mare sensibilitate o are CD3c.
Caracterizarea imunologic a LAM
MPO negative
Imunofenotiparea este esenial pentru
diagnosticul diferenial al LA MPO negative.
Expresia de CD13, CD33, MPO, cu CD19,
CD79a, CD3c i TdT negative, este fenotip de LAM.
LAM - M0 se definete n citochimia clasic avnd sub 3% blati MPO +. Prezena MPO
se poate demonstra mai bine prin imunofenotipare
i prin microscopie electronic. n aceste condiii,
imunofenotipul cu cel puin un marker mieloid pozitiv (CD113, CD33 sau MPO), n absenta markerilor limfoizi (CD3, CD22a, Cd79a), a markerilor
megakariocitari (CD41 i CD61) si eritrocitari
(CD235), permite diagnosticul de LAM - M0. Pot
fi pozitivi: HLA-DR, CD34, CD117, CD65, mai
rar CD14 i CD15 i uneori markeri limfoizi exprimai n mod aberant pe celulele mieloide (CD2,
CD4, CD7 sau CD19).
Blatii din LAM M7 exprim markeri
megakariocitari: CD41, CD42 i CD61.
n eritroleucemie blatii exprim CD235,
iar, n absena acestui marker, imunofenotipul caracteristic este CD36+/CD64-/MPO-.
Caracterizarea imunologic a celorlalte clase
morfologice de LAM
LAM-M1 i LAM-M2 se caracterizeaz

Revista Romn de Medicin de Laborator Vol. 18, Nr. 2/4, Iunie 2010

Subtypes of monocytic lineage or with

monocytic component express CD13 and CD33,
and are characterized as follows:
AML-M4 can present two blast subpopulations: monocytic cells with stronger CD45 expression and CD117-/ CD64 +, while myeloid
cells are CD117 +, CD64-;
In AML-M5 it has been proven that CD14 is
not highly specific, as blasts can be CD14 + or
negative, but CD64, CD11c, CD15, CD117 are
generally positive.
Attempts to establish morphology - immunophenotype correlations showed that, within
the same FAB classes, there are different immunophenotypes and the same immunophenotype is found in different FAB classes.
However, there are two AML classes
characterized by recurrent cytogenetic abnormalities and specific morphology, which show strong
association with certain immunophenotypes:
APL with t(15;1 7) is usually HLA-DR
and CD34 negative, with MPO + ++, CD33 +,
CD11b-, CD14-. This is particularly useful for
differential diagnosis of APL hypogranular
variant and AML M4 and M5.
AML with t (8; 21) with M2-type morphology is characterized by MPO +, CD13 +,
CD33 weakly +, HLA-DR +, CD34 + and aberrant expression of CD19, frequently asynchronous expression of CD13 and CD33,
which are weakly positive in comparison with
MPO expression.
These immunophenotypes are not considered of absolute diagnostic value but, together
with the morphology, they guide the genetic investigation towards specific recurrent abnormalities.
ALL immunophenotyping
Immunophenotyping ALL has demonstrated that many cases with FAB L3 morphology
are malignancies with mature B cells and represent
the leukemic phase of Burkitts lymphoma.
80% of cases of ALL have B precursor
immunophenotype. These blasts express a variety of B and pan-B antigens, and typically do
not express the surface immunoglobulin charac-

87

prin expresia de CD13, CD33, MPO, CD34 i

CD117, fr expresie de CD79a, CD14 i CD64.
Leucemiile de linie monocitar sau cu
component monocitar exprim CD13 i CD33,
putnd fi caracterizate n plus astfel:
n LAM-M4 se evideniaz dou subpopulaii
de blati, populaia monocitar cu expresie mai
intensa de CD45 i CD117-, CD64 +; celulele
mieloide sunt CD117+, CD64-.
n LAM-M5 s-a dovedit c CD14 nu este extrem de specific, blatii pot fi CD14+ sau
CD14-, dar CD64, CD11c, CD15 i CD117
sunt de obicei pozitive.
ncercarea de a stabili corelaii ntre
morfologie i imunofenotip, a demonstrat c, n
cadrul aceleiai clase FAB, ntlnim imunofenotipuri diferite i acelai imunofenotip se regsete n diverse clase FAB.
Totui, exist dou clase de LAM cu
anomalii citogenetice recurente i morfologie
specific, la care s-a demonstrat o corelaie puternic cu imunofenotipul, astfel:
LA promielocitar cu t(15;17) se prezint cu HLA-DR-, CD34-, MPO intens+,
CD33+, CD11b-, CD14-. Acest imunofenotip
este util n special pentru diagnosticul diferenial
dintre LA promielocitar, varianta hipogranular, i LAM - M4 sau M5.
LAM cu t(8;21) cu morfologie tip M2
este caracterizat de imunofenotipul MPO+,
CD13+, CD33 slab+, HLA-DR+, CD34+ i expresie aberant de CD19. Se ntlnete frecvent
expresia asincron de CD13 i CD33, slab pozitive comparativ cu expresia MPO.
Aceste imunofenotipuri nu reprezint un
criteriu de diagnostic, dar, mpreun cu morfologia, pot orienta investigaiile de genetic.
Imunofenotiparea LAL
Imunofenotiparea LAL a demonstrat c
multe din cazurile cu morfologie FAB L3 sunt
neoplazii cu celule B mature care reprezint faza
leucemic a limfomului Burkitt.
80% din cazurile de LAL au imunofenotip
B precursor. Aceti blati exprim o mare varietate de antigene B (tinere i pan-B) i nu exprim

88

Revista Romn de Medicin de Laborator Vol. 18, Nr. 2/4, Iunie 2010

Table 4. ALL immunophenotype

B-ALL classification according to Ig expression
PAX-5, CD19, CD20, CD22(s,cyt), CD24, CD79a (cyt), CD20 (part/dim/-), CD10 (CALLA), CD45dim, CD34, TdT
cyt

surface

Expression/ restriction of light

chain

Pro-B

Pre-T

Pre-T transitional

+ (dim)

T-ALL classification according to tymocyte maturation stage

CD2, CD3, CD4, CD5, CD7, CD8, CD1a, CD10, CD34, CD99, HLA-DR, Tdt
CD1a

CD2

cCD3

sCD3

CD4

CD5

CD7

CD8

CD34

Pre-T

+/-

+/-

Cortical

+/-

Medular

+/-

+/-

+/-

Pro-T

teristic for B mature lymphocytes. They also

contain TdT, a nuclear enzyme present in the
precursors of B or T lymphocytes but absent
from mature lymphocytes.
15% of ALL have a precursor T profile
expressing young T, pan-T antigens and TdT.
Immunological classification of ALL is important in terms of prognosis and therapeutic decision. Precursor B type leukemia has more favorable prognosis, but in this group there still are prognostic subgroups according to cytogenetic results.
ALL immunophenotype associated with
recurrent cytogenetic changes:
B-ALL with t(1, 19)(q23, p13): CD9 +, CD10
+, CD19 +, CD20-or partially +, CD34-;
B-ALL with t(4; 11)(q21, q23): CD10-, CD15
+, CD24-or partially +, CD34-;
Leukemic blasts usually differ from normal blasts by aberrant antigen expression:
asynchronous expressions (simultaneous expression of young and mature markers)
expression of antigens which are specific for
other lines
lack of expression of specific antigens
antigen overexpression.
Thus, myeloid blasts may present asynchronous expressions of CD15 (a marker of ma-

imunoglobuline de suprafa care sunt caracteristice limfocitelor B mature. De asemenea conin

TdT, enzim nuclear prezent n precursori de linie B sau T, dar absent din limfocitele mature.
15% din LAL au profil precursor T. Limfoblatii T exprim antigene T tinere, pan-T i TdT.
Clasificarea imunologic a LAL este
important din punct de vedere prognostic i al
atitudinii terapeutice. Categoria precursor B are
prognostic mai favorabil, dar i n cadrul acestui
grup exist subgrupe de prognostic, n funcie de
rezultatele citogenetice.
Imunofenotipurile LAL asociate cu modificri citogenetice recurente sunt:
B-LAL cu t(1;19)(q23;p13), imunofenotip: CD9+,
CD10+, Cd19+, CD20- sau parial +, CD34 B-LAL cu t(4;11)(q21;q23), imunofenotip
CD10-, CD15+, CD24- sau parial +, CD34-.
Blatii din LA se deosebesc de obicei de
blatii din hematopoieza normal prin expresii
aberante de antigene:
expresii asincrone (expresie concomitent de
markeri de celul tnr i matur);
expresia de antigene specifice pentru alte linii;
lipsa expresiei unor antigene specifice;
supraexpresia unor antigene.
Astfel, blatii mieloizi pot prezenta ex-

Revista Romn de Medicin de Laborator Vol. 18, Nr. 2/4, Iunie 2010

89

Table 5. EGIL scoring for biphenotypic leukemia

Lineage
Scoring

myeloid

CD79
c

CD3
TCR

MPO(lysozime)

CD 2
CD19
CD10
CD20

CD2
CD5
CD8
CD10

CD13
CD33
CDw65
CD117

0.5

TdT
CD24

TdT
CD17
CD1a

CD24
CD25
CD64

turity) and CD117 (a marker usually present on

young elements of the myeloid lineage) and the
expression of antigens specific for other lines:
CD19, CD7, CD56
Lymphoblasts can express aberrant myeloid antigens, B lymphoblasts sometimes express CD20 and T lymphoblasts may express
asynchronous markers.
It is important to establish the leukemiaassociated immunophenotype for each individual case at diagnosis. This may be useful later in
the follow-up and the diagnosis of minimal residual disease, with the specification that blasts
may undergo immunophenotypic changes during evolution or post-therapy.
Diagnosis of acute leukemia with mixed
phenotype is the result of immunophenotyping
and applying the EGIL score. This score assigns
values of 2, 1, 0.5 points for markers of B, T and
myeloid lineage, respectively (Table 5).
AL with mixed phenotype is further
classified according to recurrent cytogenetic abnormalities seen in this group.

The role of cytogenetics in AL

Clonal cytogenetic abnormalities, both
numerical and structural, are present in 60-80%
of the AML and respectively 80% of the ALL.
Identification of genetic changes in AL
at karyotype and molecular level allowed for:

presii asincrone CD15 (marker de maturitate) cu

CD117 (marker prezent de obicei pe elemente tinere) i expresia de antigene specifice pentru
alte linii: CD19, CD7 sau CD56.
Limfoblatii pot exprima n mod aberant
antigene mieloide, limfoblatii B exprim uneori
CD20, iar limfoblatii de linie T exprim uneori
markeri asincroni.
Pentru fiecare caz n parte este important stabilirea la diagnostic a imunofenotipului
asociat leucemiei. Acesta poate fi util ulterior
pentru urmrirea n dinamic i pentru diagnosticul de boal minim rezidual, cu specificaia
c imunofenotipul blatilor poate suferi modificri pe parcursul evoluiei sau post-terapie.
Diagnosticul de leucemie acuta cu fenotip
mixt se stabileste pornind de la imunofenotipare.
Scorul EGIL atribuie valori de 2, 1, 0.5 puncte pentru markerii de linie B, T i mieloizi (Tabelul 5).
Scorul peste 2 pentru dou din linii definete LA
bifenotipic. Clasificarea OMS 2008 redefinete
criteriile pentru LA de linie ambigu, incluznd LA
bifenotipice si biliniale n aceast categorie.
LA cu fenotip mixt sunt clasificate n
continuare n funcie de anomaliile citogenetice
recurente ntlnite la acest grup.

Rolul citogeneticii n LA
Anomalii citogenetice clonale, att numerice ct i structurale, se ntlnesc la 60-80%

90

classification

Revista Romn de Medicin de Laborator Vol. 18, Nr. 2/4, Iunie 2010

into prognostic classes- cytogenetic changes were found to be significant independent prognostic factor, with different recommendations regarding the usefulness and
right moment for bone marrow transplantation;
a better understanding of the leukemogenesis
with achievements and promises regarding
drug therapy.
In practice, standard cytogenetic analysis of chromosome G banding is still the most
used method to detect many rearrangements in
the leukemic clone. It is a laborious method, performed by trained personnel, requires the presence of dividing cells in the analysis sample, examines a small number of metaphases and leaves
no evidence of submicroscopic mutations.
Molecular biology techniques currently
used in the diagnosis and classification of LA are
fluorescent in situ hybridization (FISH) and
polymerase chain reaction (PCR) used for:
clonality demonstration (e.g. TCR - T cell receptor rearrangements);
identification of cryptic mutations in classic cytogenetics, i.e. mutations involving small fragments;
explaining complex caryotypes.
These investigations are guided by clinical,
morphological and cytogenetic aspects. Molecular
biology finds its practical application in AL, when
contributing to diagnosis (see AL with recurrent
cytogenetic abnormalities), or documents a significant abnormality in terms of prognosis.
PCR technique also tends to find its
place in the definition of complete remission and
minimal residual disease for many types of rearrangements, provided the methods used are
standardized.
Fluorescent in situ hybridization (Fluorescent in Situ Hybridization) combines the advantages of standard cytogenetic analysis with
molecular techniques. It allows identification of
clonal rearrangements in metaphase and interphase nuclei and analyzes more cells than
conventional cytogenetics.
Recommended PCR techniques use
RNA as target and synthesize by reverse-transcription a DNA copy subsequently amplified

din LAM i respectiv la 80% din LAL.

Identificarea modificrilor genetice din LA,
la nivelul cariotipului i la nivel molecular, a permis:
ncadrarea LA n clase de risc - modificrile citogenetice s-au dovedit a fi factor prognostic
major i independent, cu recomandri diferite
n ceea ce privete utilitatea i momentul optim
al transplantului medular;
o mai buna nelegere a fenomenului leucemogenezei cu perspective n ceea ce privete terapiile medicamentoase.
n practic, analiza citogenetic standard cu bandare G a cromozomilor este nc
metoda cea mai utilizat pentru a detecta ct mai
multe dintre rearanjamentele de la nivelul clonei
leucemice. Aceast tehnic necesit prezena de
celule n diviziune n proba de analizat, analizeaz un numr mic de metafaze i nu permite evidenierea mutaiilor submicroscopice.
Tehnicile de biologie molecular utilizate n prezent pentru diagnosticul i clasificarea
LA sunt hibridizarea fluorescent in situ
(FISH) i reacia de polimerizare n lan
(PCR). Acestea se utilizeaz pentru:
demonstrarea clonalitii (de exemplu rearanjamente TCR T cell receptor);
identificarea mutaiilor criptice, adic acele mutaii
care implic fragmente mici, submicroscopice;
descifrarea cariotipurilor complexe.
Conducerea acestor investigaii este orientat de aspecte clinice, morfologice, citogenetice.
Rolul biologiei moleculare este de a contribui la
diagnostic (vezi LA cu anomalii citogenetice recurente) i de a demonstra prezena unor anomalii importante din punct de vedere prognostic.
De asemenea, se tinde ca tehnica PCR
s i gseasc locul n definirea noiunilor de
remisiune complet i boal minim rezidual
pentru ct mai multe tipuri de rearanjamente, cu
condiia standardizrii metodelor utilizate.
Hibridizarea fluorescent in situ combin avantajele analizei citogenetice standard cu
tehnicile moleculare. Aceasta permite vizualizarea rearanjamentelor pe celule n metafaz i n
interfaz i analiza unui numr mai mare de celule dect citogenetica clasic.

Revista Romn de Medicin de Laborator Vol. 18, Nr. 2/4, Iunie 2010

91

Table 6 Cytogenetic prognosis classes in AML

Favorable

Intermediate

Unfavorable

Inv (16) , t(16;16)(p13;q22), del(16q)

t(8;21)(q22;q22)
t(15;17)
Abnormalities of 11q 23 ex. t(11 ;19)(q23 ;p13.1)
+8, +21, +22, +11,+13, +6
t(6;9)
del(5q), del(7q), del(9q), del(11q), del(12p), del(20q)
-7, -y
-5, -7
t(v;11)(v;q23)
Inv(3), t(3 ;3)
Complex karyotype (>= 3 unrelated abnormalities)
t(6 ;9), t(6 ;11), t(11 ;19)

(RT-PCR). DNA is recommended as target only

when break points are located nearby, on a short
fragment of DNA.

Important genetic rearrangements in AML

Translocation (15;17)(q22;q21) is always
associated with APL, a subtype of AML with clinical
and morphological distinctive characters. The fusion
product of the PML gene on chromosome 15 with
RAR-alpha gene on chromosome 17 triggers a pathway to block granulocytic maturation at the promyelocytic stage. This is a pathogenetic mechanism on
which the therapy with ATRA (All Trans Retinoic
Acid) specifically acts. No other AML has a therapeutic response to ATRA. There are molecular variants of APL which do not respond to ATRA therapy.
AML with rearrangements involving CBF
(Core binding factor)
t(8;21)(q22;q22) leads to the fusion gene
of AML1 (CBF-alpha) gene on chromosome 21
with the ETO (eight twenty one) on chromosome
8. 90% are AML-M2 with Auer rods, dysgranulopoiesis, eosinophils in large numbers in bone
marrow, and favorable response to repeated
cycles of high dose cytarabine.
Inv (16) or t(16;16)(p13;q22) (CBF-betaMYH11) have M4Eo morphology with high percentage of immature eosinophils with primary
(basophilic) granules and a favorable prognosis.

Tehnicile preferate de PCR sunt cele care utilizeaz ARN-ul ca int i sintetizeaz sub aciunea revers-transcriptazei (RT) o copie de ADN, care ulterior
se amplific (RT-PCR). ADN este recomandat ca int
doar pentru situaiile n care punctele de ruptur sunt
situate n apropiere, pe un fragment scurt de ADN.

Rearanjamente genetice importante n LAM

Translocaia (15;17)(q22;q21) se asociaz ntotdeauna cu LAP, subtip de LAM cu caractere clinice i morfologice distincte. Produsul
de fuziune a genei PML de pe cromozomul 15
cu gena RAR- de pe cromozomul 17 declaneaz o cale de blocare a maturaiei pe linia granulocitar la nivel de promielocit, mecanism patogenetic asupra cruia acioneaz n mod specific
terapia cu ATRA (all trans retinoic acid). Nici o
alt LAM nu beneficiaz de aceast terapie.
Exist variante moleculare de LAP care nu rspund la terapia cu ATRA.
LAM cu rearanjamente ce implic CBF (Core
binding factor)
t(8;21)(q22;q22) se soldeaz cu fuziunea
genei AML1(CBF-) de pe cromozomul 21 cu
gena ETO (eight twenty one) de pe cromozomul 8.
O proporie important (90%) sunt LAM-M2 cu
corpi Auer, disgranulopoiez, eozinofile n numr
mare n mduv, i rspuns favorabil la cicluri repetate cu citarabin n doz mare.

92

Revista Romn de Medicin de Laborator Vol. 18, Nr. 2/4, Iunie 2010

AML with 11q23 abnormalities involving the MLL gene: the most common is
t(9;11)(q23;q23) (MLL-AF9), often with
monoblastic morphology and unfavorable prognosis. Mutations are cryptic for classical cytogenetics and are identified by RT-PCR or FISH.
Other common cytogenetic abnormalities
in AML are trisomy 8, monosomy 5, 7, del(5q),
del (7q); there are also AML with normal karyotype and AML with complex karyotype.

Cytogenetic abnormalities in LAL

The most common cytogenetic abnormality
in precursor B ALL is hyperdyploidy with 50 chromosomes, most frequently encountered in patients of
2-10 years of age, associated with low or intermediate number of blasts in peripheral blood. An additional copy of chromosomes 4 and/or 10 is frequently
encountered. ALL with 50 chromosomes hyperdyploidy has a very good therapeutic response.
Structural cytogenetic changes in B
ALL have an intermediate to unfavorable prognosis. An exception is t(12;21)(p12;q22),
which is usually not showed by classical cytogenetics. It requires FISH or PCR for identification and has a favorable prognosis.
Patients with precursor B ALL with t(9;22)
(q24;q11) or abnormalities on 11q23- most commonly t(4;11)(q21;q23), have unfavorable prognosis.
Strategy for genetic investigation of
acute leukemia may vary, but always involves a
cytogenetic analysis at onset, followed if necessary by molecular biology studies, for example:
for ALL B cases in children one should seek
cryptic translocation t(12;21)(p13;q22) ETV6RUNX1 bu using FISH or PCR;
cases of AML with morphology suggestive for
inv(16) or t(16;16)(p13.1;q22) should be confirmed by genetic analysis;
in cases of Burkitts lymphoma, in which
MYC gene translocation from chromosome 8
to chromosome 14 specifically occurs, the
PCR technique is not recommended due to the

LA cu inv (16) sau t(16;16)(p13;q22)

(CBF- - MYH11) au morfologie M4Eo cu procent mare de eozinofile imature cu granulaii
primare (bazofile) i prognostic favorabil.
LAM cu anomalii 11q23 cu implicarea genei MLL
Cea mai frecvent este t(9;11)(q23;q23)
(MLL-AF9), deseori cu morfologie monoblastic i prognostic defavorabil. Mutaiile sunt criptice i se identific prin RT-PCR sau FISH.
Alte anomalii citogenetice ntlnite n
LAM sunt trisomia 8, monosomia 5, 7, del(5q),
del(7q); se descriu de asemenea LAM cu cariotip normal sau complex.

Anomalii citogenetice n LAL

Cea mai comun anomalie citogenetic
n LAL precursor B este hiperdiploidia cu 50
de cromozomi. ntlnit cel mai frecvent la pacieni de 2-10 ani, se asociaz cu numr mic sau
intermediar de blati n sngele periferic. Frecvent se ntlnete o copie n plus pentru cromozomii 4 i/sau 10. LAL cu hiperdiploidie cu 50
de cromozomi au rspuns terapeutic foarte bun.
Modificrile citogenetice structurale n
LAL B au prognostic intermediar sau defavorabil. O excepie o reprezint t(12;21)(p12;q22)
care nu este evideniabil de obicei prin citogenetica clasic i trebuie identificat prin FISH
sau PCR, cu prognostic favorabil.
Pacienii cu LAL precursor B cu t(9;22)
(q24; q11) sau anomalii la nivelul 11q23 - mai
frecvent translocaia t(4;11)(q21; q23), au prognostic defavorabil.
Strategia de investigare genetic a
leucemiilor acute poate s difere, dar presupune
ntotdeauna o analiz citogenetic la debut, urmat, n funcie de caz, de studii de biologie molecular, de exemplu:
pentru cazurile de LAL B la copii se caut
translocaia criptic t(12;21)(p13;q22) ETV6RUNX1 prin FISH sau PCR;
cazurile de LAM cu morfologie sugestiv pentru inv(16) sau t(16;16)(p13.1;q22) vor fi investigate n acest sens;

Revista Romn de Medicin de Laborator Vol. 18, Nr. 2/4, Iunie 2010

heterogeneity of break points and, as such,

FISH is preferred;
identification of specific translocations for
APL in cases with uncommon morphology is
essential for initiating treatment with ATRA
and reduces the risk of DIC, extremely high in
these cases;
identification of BCR-ABL fusion gene (p190)
with prognostic significance in ALL.
Current theory on leukemogenesis implies the existence and time sequence of at least
two mutations: class I mutation providing survival and proliferation advantage and class II
mutation affecting the differentiation, maturation and apoptosis.
Thus NPM1, FLT3-TKD and CEBP are
class II mutations with favorable prognosis, unless
FLT3-ITD mutation is associated. The latter is a
class I mutation with unfavorable prognosis.
Other mutations with unfavorable prognosis are MLL-PTD (class II), overexpression
of BAALC gene (class II), ERG overexpression
(class I), and WT1 mutation.
In conclusion, the diagnostic steps for
LA currently involve conducting the following
investigations:
automated blood cell count and blood film
morphology
bone marrow aspiration biopsy- is absolutely
necessary until the number of blasts in peripheral blood is high
cytochemistry and immunophenotyping by
flow cytometry
cytogenetic examination in all cases, RT-PCR
and FISH for selected cases.
Other laboratory investigations essential
to describe the clinical-biological status of the
patient at onset and in follow-up are: coagulation studies, biochemical determinations: LDH
and uric acid are usually high in patients with
LA, liver and kidney function assessment before
starting treatment, microbiologic investigation
of different biological products for appropriate
antimicrobial therapy, HLA and DNA typing for
potential candidates for allogenic transplant.

93

pentru cazurile de limfom Burkitt, n care n mod

specific se produce translocaia genei MYC de

pe cromozomul 8 pe cromozomul 14, tehnica
PCR nu este recomandat datorit heterogenitii
punctelor de ruptur i se prefer FISH;
identificarea translocaiei specifice pentru LAP n cazurile cu morfologie necaracteristic este esenial
pentru iniierea tratamentului cu ATRA i reducerea
riscului de CID, extrem de mare la aceste cazuri;
identificarea genei de fuziune ABL-BCR
(p190) cu importan prognostic n LAL.
Teoria actual a leucemogenezei presupune existena i succesiunea n timp a cel puin
dou mutaii: o mutaie de clasa I, care ofer
avantaj de supravieuire i proliferare a clonei i
o mutaie de clasa II, care altereaz diferenierea, maturaia i apoptoza.
Astfel mutaiile NPM1, FLT3-TKD i
CEBP - sunt mutaii de clasa II cu prognostic
favorabil, cu condiia s nu se asocieze cu mutaia FLT3-ITD. Aceasta din urm este o mutaie
de clas I cu prognostic defavorabil.
Alte mutaii cu prognostic defavorabil sunt
MLL-PTD (clasa II), supraexpresia genei BAALC
(clasa II), supraexpresia ERG (clasa I), mutaia WT1.
In concluzie, etapele de diagnostic al LA
presupun n prezent efectuarea urmtoarelor investigaii:
hemograma, frotiu sanguin
puncie - biopsie medular - se poate renuna
doar dac numrul de blati din sngele periferic este mare;
citochimie sau imunofenotipare prin citometrie
n flux;
citogenetic pentru toate cazurile, RT-PCR i
FISH pentru cazuri selectatate.
Alte investigaii indispensabile pentru conturarea tabloului clinico-biologic al bolnavului cu LA
la debut i n evoluie sunt explorarea coagulrii, determinri biochimice (LDH i acid uric sunt de obicei
mari la pacienii cu LA), explorarea funciei hepatice
i renale nainte de iniierea tratamentului, culturi din
produse biologice pentru terapie antimicrobian
adecvat, tipizare HLA i ADN pentru pacienii poteniali candidai pentru transplant allogen.

94

Revista Romn de Medicin de Laborator Vol. 18, Nr. 2/4, Iunie 2010

LAM M1

LAM M1 peroxidaza

Corpi Auer

LAM M2 peroxidaza

LAM M3

LAM M3 varianta

Revista Romn de Medicin de Laborator Vol. 18, Nr. 2/4, Iunie 2010

LAM M4

LAM M4 cu eozinofile

LAM M55

LAM M6

LAL 1/ LAL 2

LAL 3

95

96

Revista Romn de Medicin de Laborator Vol. 18, Nr. 2/4, Iunie 2010

Abbreviations:

Abrevieri:

ABL BCR Abelson - breakpoint cluster region,

AL acute leukemia,
ALL acute lymphoblastic leukemia,
AML acute myeloid leukemia,
APL acute promyelocytic leukemia,
ATRA all trans retinoic acid,
BAALC Brain and Acute Leukemia, Cytoplasmic,
CBF core binding factor,
CD cluster of differentiation,
CEBPA CCAAT/enhancer-binding protein,
del deletion,
DIC disseminated intravascular coagulation,
DNA deoxyribonucleic acid,
EGIL European Group for the Immunological
Characterization of Leukemias,
ERG Ets -erythroblastosis virus E26 oncogene
homolog - related gene,
FAB French - American - British,
FISH fluorescent in situ hybridization,
FLT3 FMS-like tyrosine kinase 3,
HLA human leukocyte antigen,
i inversion,
ITD internal tandem duplication,
M4Eo acute myeloid leukemia M4 with
eosinophilia,
MDS myelodysplastic syndrome,
MGG May Grunwald Giemsa,
MIC morphology, immunophenotyping,
cytogenetics,
MLL mixed lineage leukemia,
MPO or POX Myeloperoxidase,
MYH myosine heavy chain,
NPM1 nucleophosmin 1,
PCR polymerase chain reaction,
PTD partial tandem duplication,
RNA ribonucleic acid.
RT-PCR reverse transcriptase polymerase chain
reaction,
SSC side scatter,
t translocation,
TCR T-cell receptor,
TdT Terminal deoxynucleotidyl transferase,
TKD tyrosine kinase domain,
WHO World Health Organization,
WT Wilms tumour,

ABL-BCR Abelson - breakpoint cluster region,

ADN acid dezoxiribonucleic,
ARN acid ribonucleic
ATRA all transretinoic acid,
BAALC Brain and Acute Leukemia, Cytoplasmic,
CBF core binding factor,
CD cluster of differentiation,
CEBPA CCAAT/enhancer-binding protein,
CID coagulare intravascular diseminat,
del deleie,
EGIL European Group for the Immunological
Characterization of Leukemias,
ERG Ets -erythroblastosis virus E26 oncogene
homolog - related gene,
FAB French - American British,
FISH fluorescent in situ hybridization,
FLT3 FMS-like tyrosine kinase 3,
HLA human leukocyte antigen,
i inversiune,
ITD internal tandem duplication,
LA leucemie acut,
LAL leucemie acut limfoblastic,
LAM leucemie acut mieloid,
LAP leucemie acut promielocitar,
M4Eo leucemia acut mieloid M4 cu eozinofile,
MGG May Grunwald Giemsa,
MIC morphology, immunophenotyping, cytogenetics,
MLL mixed lineage leukemia,
MPO mieloperoxidaz,
MYH myosine heavy chaine,
NPM1 nucleophosmin 1,
OMS Organizatia Mondial a Sntaii,
PCR polymerase chain reaction,
PTD partial tandem duplication,
RT-PCR reverse transcriptase polymerase chain
reaction,
SMD sindrom mielodisplazic,
SSC side scatter,
t translocaie,
TCR T-cell receptor,
TdT Terminal deoxynucleotidyl transferase,
TKD tyrosine kinase domain,
WT Wilms tumour.

Revista Romn de Medicin de Laborator Vol. 18, Nr. 2/4, Iunie 2010

97

References

References

1. Paiu M. Examenul citologic al frotiului sangvin. Editura Alma Mater, Cluj-Napoca 2009
2. Cucuianu A. Leucemiile acute. In Hematologie cliniceditor coordonator Petrov L. Casa Crii de tiin. ClujNapoca 2009
3. Vardiman JW, Thiele J, Arber DA, Brunning RD,
Borowitz MJ, Porwit A, Harris NL, Le Beau MM, Hellstrm-Lindberg E, Tefferi A, Bloomfield CD. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale
and important changes. Blood 2009 114: 937-951
4. Amy Heerema-McKenney, Arber DA. Acute Myeloid
Leukemia. Hematol Oncol Clin N Am 23 2009: 633654
5. Onciu M. Acute Lymphoblastic Leukemia. Hematol
Oncol Clin N Am 2009 114: 937-951
6. Dhner H, Estey EH, Amadori S, Appelbaum FR,
Bchner T, Burnett AK, Dombret H, et al; European LeukemiaNet. Diagnosis and management of acute myeloid
leukemia in adults: recommendations from an international
expert panel, on behalf of the European LeukemiaNet.
Blood 2010 115(3):453-74
7. Fiona E. Craig, Foon KA. Flow cytometric immunophenotyping for hematologic neoplasm. Blood 2008 111:
3941-3967
8. Marie C. Bn. Biphenotypic, bilineal, ambiguous or
mixed lineage: strange leukemias! Haematologica 2009
94(7): 891-893
9. Frattini MG, Maslak PG. Strategy for Incorporating
Molecular and Cytogenetic Markers into Acute Myeloid
Leukemia Therapy. J. Natl. Compr. Canc. Netw. 2008
6(10):995-1002

1. Paiu M. Examenul citologic al frotiului sangvin

Editura Alma Mater, Cluj- Napoca 2009
2. Cucuianu A. Leucemiile acute. In Hematologie cliniceditor coordonator Petrov L. Casa Crii de tiin ClujNapoca 2009
3. Vardiman JW, Thiele J, Arber DA, Brunning RD,
Borowitz MJ, Porwit A, Harris NL, Le Beau MM,
Hellstrm-Lindberg E, Tefferi A, Bloomfield CD. The 2008
revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood 2009 114: 937-951
4. Amy Heerema-McKenney, Arber DA. Acute Myeloid
Leukemia. Hematol Oncol Clin N Am 23 2009: 633654
5. Onciu M. Acute Lymphoblastic Leukemia. Hematol
Oncol Clin N Am 23 2009 ;114: 937-951
6. Dhner H, Estey EH, Amadori S, Appelbaum FR,
Bchner T, Burnett AK, et al; European LeukemiaNet. Diagnosis and management of acute myeloid leukemia in
adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet. Blood 2010
Jan 21;115(3):453-74
7. Fiona E. Craig, Foon KA.
Flow cytometric
immunophenotyping for hematologic neoplasm. Blood
2008; 111: 3941-3967
8. Marie C. Bn. Biphenotypic, bilineal, ambiguous or
mixed lineage: strange leukemias! Haematologica;2009;
94(7) : 891-893
9. Frattini MG, Maslak PG. Strategy for Incorporating
Molecular and Cytogenetic Markers into Acute Myeloid
Leukemia Therapy. J. Natl. Compr. Canc. Netw. 2008
6(10):995-1002